COMPOSITIONS AND METHODS FOR TRANSDERMAL DRUG DELIVERY

Abstract: The present invention relates to methods of delivering nucleotide-based pharmaceutical agents across membranes, such as the dermis or skin layer of a patient using iontophoresis.

Description: COMPOSITION^ ΛND METHOD^ FOR TR WSDERMA1 DRUG Dili I\TR\ CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of application serial No 08/ 107,329, tiled August 16, 1993, which is continuation-in part ot application Serial No 08/077,786, filed June 14, 1993, which is a continuation-in-part of U.S. application Serial No. 08/009,463, filed lanuarv 27, 1993 This application is also a contmuation-in-part of U.S. application Serial No. 08/077,296, filed )une 14, 1993 All ot the above identified applications are incorporated herein by reference tor all purposes BACKGROUND OF THE INVENTION The present invention relates generallv to methods of delivering pharmaceutical agents across membranes, such as the dermis or skin layer ot a patient More particularly, the invention relates to methods tor lontophoreticailv delivering nucleotide-based pharmaceutical agents Heretofore, nucleotide-based drugs, such as aptamers, πbozvmes, antisense compounds, and triple helix drugs have had limited success as therapeutic agents, in part, because ot problems associated with their stability and delivery Nucleotide-based pharmaceutical agents contain a phosphodiester group which is sensitive to degradation bv nucleases. Such degradation would be a significant impediment to the use ot an oiigonucieohde or nucleic acid as a pharmaceutical agent that depends upon the integrity ot the sequence tor its recognition specificity. Thus, naturally occurring oligonucieotides and nucleic acids often must be chemically modified to render them resistant to nucleases which would degrade them in vivo, or even in vitro unless care is taken to choose appropriate conditions. The therapeutic efficacy ot pharmaceutical or therapeutic agents, including nucleotide-based pharmaceuhcal agents, relies on the delivery ot adequate doses ot a pharmaceutical agent to the site of action Many modes ot delivery have been developed which include, tor example, enteral (oral), parenteral (intramuscular, intravenous, subcutaneous ), and topical administration In most instances, the administration svstem is chosen tor reliable dosace delivery and convenience Typically, parenteral administration is the most reliable means of delι\ eπnc a pnarmaceutical to a patient See υυdman i t al , Goodman and Gilman s Pharmacological Basis of Therapeutics, Pergamon Press, Elmsrord. New Y ork (1990) and Pratt ct al Principles of Drug Action The Basis ot Pharmacoio . . Churchill Livingstone, New York, New York ( 1990) Each parenteral mecnanism insures that a prescribed dosage ot the pharmaceutical agent is inserted into the fluid compartment ot the bodv wnere it can be transported. The disadvantage ot these modes of delivery is that thev require an invasive procedure The invasive nature of administration is inconvenient, painful, and subject to infectious contamination Enteral and topical administration are more convenient, generally non-painful, and do not predispose to infection; however, both are limited. The gastrointestinal and dermal surfaces present formidable barriers to transport, and therefore, some pharmaceutical agents are not absorbed across these surfaces. Another drawback to patient directed modes ot administration (enteral, topical and subcutaneous) is compliance. Pharmaceutical agents which have a short half- life require multiple daily doses As the number of doses increases, patient compliance and therapeutic efficacy decrease. Simplified and/or infrequent administration schedules would aid in optimizing patient compliance. Wilson et al. (1991) Harrison's Principles of Internal Medfcine. 12th Ed., McGraw-Hill, Inc., New York, New York. Efforts to develop more effective and convenient modes ot pharmaceutical administration have led to the development ot transdermal delivery systems. Many current transdermal pharmaceutical agent delivery systems rely upon pharmaceutical agents which are absorbed when admixed with inert carriers. See Cooper et al. (1987) "Penetration Enhancers", in Transdermal Delivery of Drugs. Vol II, Kvodonieus et al , Eds.. CRC Press, Boca Raton, Florida. Few pharmaceutical agents tit this profile and those which do are not always predictably absorbed. In addition, the skin is an efficient barrier to the penetration ot water soluble substances, and the rate ot transdermal pharmaceutical agent absorption is determined bv iipid solubility and polarity. Highly polar or water soluble pharmaceutical agents are effectively blocked. Even very lipophihc pharmaceutical agents penetrate the dermis verv slowly compared with the rate of penetration across cell membranes See Pratt et al. supra. Various forms ot chemical enhancers, such as those enhancing iipophiliαtv, have been developed to improve transdermal transport when physically mixed with certain therapeutic agents and provide more predictable absorption See tor example, U.S. Patents 4,645,502, 4,788,062. 4,816,258, 4,900.555, 3,472.931, 4,006.218. and 5,053,227. Carriers have also been coupled to pharmaceutical agents to ennance intracellular transport See Ames ct al (1973) Proc Natl. Acad. Sci USA. 70.456-458 and (1988) Proc int Svmp Cont Rel Bioact Mater., 15 142 Electric gradients also have been used to enhance transdermal pharmaceutical agent delivery. See Chien ct al. (1989) journal of Pharmaceutical Sciences, 78(5):353-354 and Banga ct al. ( 1988) I Controlled Release, 7.1-14. This technique, known as iontophoresis, uses electrostatic forces to enhance the rate ot delivery ot ionized pharmaceutical agents through the skin. For lontophoretic delivery, the drug molecules must be in ionized state with either a positive or negative charge. The rate ot drug delivery in iontophoresis is directly propornonal to the svstem current; the higher the current, the greater the driving force and pharmaceutical agent delivery. Typically, devices are used which hold a pharmaceutical agent in a reservoir near the skin, generate an electric field surrounding the pharmaceutical agent-dermal interface, and drive the agent through the skin. Ionic strength also affects the lontophoretic drug delivery rate. See Banga supra. Ionic strength is related to the concentration ot various ions present in the solution of the pharmaceutical agent in the reservoir. The greater the ionic strength (i.e., the more ionized particles per unit volume), the higher the concentration of ions, and the greater the competition for the electric current. Other factors which mav affect the delivery rate include pH, concentration, extraneous ions (e.g., from buffer solutions), conductivity, and electronic factors. Electroporation has been used as a method of delivering nucieic acids. In this method, one or more pulses of electrical energy of sufficient voltage and duration to produce a transient increase in Ussue permeability is applied to a region of a tissue. A driving force, for example an electrical, physical, or chemical force, such as provided bv a temperature gradient, a pressure gradient, a concentration gradient, or an acoustic or optical pressure, is then utilized to move the molecules across the permeabihzed tissue. Because ot the potential therapeutic benefits associated with nucleotide-based drugs and the difficulties associated with their delivery, there exists a significant need for improved methods for delivering these pharmaceutical agents in a controlled fashion and particularly, for delivery methods which improve the bioavailability or other pharmacological properties of these pharmaceutical agents. The present invention fulfills these needs. SUMMARY OF THE INVENTION The invention provides methods tor delivering a nucleotide-based pharmaceutical agent in a therapeuticallv ettective dose to a patient, the method comprising the steps ot. contacting the skin of a patient with a theraDeuticallv effective amount ot a nucleotide-based pharmaceutical agent in an lontophoretic device, wherein the pharmaceutical agent is lorucaiiv charged; and applying an electric field to the interface of the lontophoretic device and the skin, such that the pharmaceutical agent is delivered transdermaih The compositions and methods described herein can De utilized tor either the svstemic or local administration ot a nucleotide-based pharmaceutical agent, depending on the therapeutic indication. According to a preferred embodiment of this invention, an electric field between about 0.1 and 0.5 mAmp/cm**- is applied to deliver a nucleotide-based pharmaceutical agent in an iontophoresis device. This embodiment results in the deliverv of between about 0.1 and 20 mg, and preferablv 0.1 and 10 mg ot the pharmaceutical agent during a 24 hour period for an iontophoresis device having about a 20 cm^ donor reservoir. The nucleotide-based pharmaceutical agents that can be delivered using the methods described herein will generally comprise oligonucleotides and nucleic acids, having between about 2 and 100 nucleotides, preferablv between about 2 and 50 nucieotides, and more preferablv between about 10 and 40 nucleotides. Representative pharmaceutical agents include aptamers, πbozvmes, antisense compounds, and triple helix drugs. According to some embodiments, the nucleotide-based pharmaceutical agent is contacted with a hposomal formulation, such as Lipofectin™ prior to delivery of the pharmaceutical agent. BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a graphic depiction ot the effect of various concentrations of sodium chloride on flux (nanograms (ng)/cm^-hr) as a function ot time (hours). Fig. 2 is a graphic depiction of the effect of ohgonucleotide size on flux (ng/cm-'-hr) as a function ot time (hours). Fig. 3 is a graphic depiction ot the effect ot ohgonucleotide concentration on flux (ng/cm^-hr) as a function of time (hours). Fig. 4 is a graphic depiction of the effect of Lipofectin™ concentration on flux (ng/cm-^-hr) as a function of time (hours ) Fig. 5 is an illustration ot the structure ot the thrombin aptamer d(GGTTGGTGTGGTTGG). Fig. b is a graphic depiction of the effect ot ohgonucleotide conformation on flux (ng/ cm*--hr) as a function ot time ( hours ) DETAILED DESCRIPTION OF THE INVENTION CONTENTS I Terminology II lontophoretic Delivery of Nucieotide-Based Drugs A. Overview B. Aptamers C Ribozymes D Antisense Compounds E. Triple Helix Drugs F. Analogs and Derivatives G. Secondary Structure III. Compositions of Nucleotide-based Pharmaceuticals IV. In Vitro Testing of Nucieotide-Based Pharmaceutical Agents V. In Vivo De verv of Nucieotide-Based Pharmaceutical Agents A. Therapeutic Indications B. Dosages C. lontophoretic Delivery i. Terπuno.ggy The following terms are intended to have the following general meanings: "Pharmaceutical agent or drug " refers to anv chemical or biological material, compound, or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Some drugs are sold in an inactive form that is converted in vivo into a metabolite with pharmaceutical activitv. For purposes of the present invention, the terms "pharmaceutical agent" and "drug" encompass both the inactive drug and the active metabolite. "Transdermal delivery " refers to the transport of substance across the epidermis and dermis, such as the skin or mucous membranes, where the substance can contact and be absorbed into the capillaries. In certain instances, the delivery will be enhanced across other membranes. "Enhanced transdermal delivery " refers both to the facilitation of transdermal delivery and an absolute increase in the molar volume transported per unit time through a constant surface area utilizing an equimoiar pool of transported material as compared to unenhanced transdermal delivery Iontophoresis ' or lontophoretic ' refers to the introduction ot an lonizable chemical through skin or mucous membranes bv the application ot an electric field to the interface between the lonizaole chemical compound and the skin or mucous membrane 'Permeability' refers to the ability of an agent or substance to penetrate, pervade, or diffuse through a barrier, membrane, or a skin laver 5 "Pharmaceutically or therapeuticallv effective dose or amount refers to a dosage level sufficient to induce a desired biological result That result can be transdermal de verv ot a pharmaceutical agent, alleviation of the signs, symptoms, or causes ot a disease, or any other desired alteration ot a biological svstem. 1 0 "Nucleotide" refers to a phosphoric acid ester ot a N-glycoside of a heterocyclic nitrogenous base and is meant to encompass both non-cyclic and cyclic derivatives The phosphate can be present on position 2', 3', and/or 5'. Generally, the giycoside component will be a pentose, however, in some embodiments, hexoses will be employed. The nitrogenous base typically will be selected from the 1 5 g rou P consisting ot adenine, guarune, hvpoxanthine, uracil, cvtosme, and thyπune, and analogs or chemical modifications thereof "Nucleotide-based pharmaceutical agent" or nucleotide-based drug ' refer to a pharmaceutical agent or drug comprising an ohgonucleotide or nucleic acid. 2 0 "Ohgonucleotide" generally refers to linear sequences of nucleotides, joined by phosphodiester bonds, typically prepared bv synthetic means. Position 3' of each nucleotide unit is linked via a phosphate group to position 5' of the next unit. In the terminal units, the respective 3' and 5' positions can be free (I e., free hvdroxvi groups) or phosphorvlated. Those ohgonucleotides employed in the 2 5 present invention will varv widely in length, generally from 2 to 100 nucleotides Ohgonucleotides and nucleic acids can De described in terms ot their length. For example, an ohgonucleotide comprising 20 nucleotides is termed a "20-mer , whereas an ohgonucleotide having 30 nucieotides is a "30-mer ' Suitable ohgonucleotides can be prepared bv the phosphoramidite 3 0 method described by Beaucage and Carruthers, Tetra. Letts. 22:1859-1862 (1981), or by the tπester method according to Matteucci, et al., J. Am. Chem. Soc, 103:3185 (1981), or by other methods, such as commercial automated ohgonucleotide synthesizers. Ohgonucleotide also is meant to include chemical modifications ot the naturally occurring ohgonucleotide skeleton Such modifications include, but ~ , 5 are not limited to, modifications at cvtosine exoevche amines, substitution ot 5- bromouracil, backbone modifications, base analogs, methvlations, and the like Infra "Nucleic acid refers to either deoxvπbonucleic acid (DNA) or πbonucieic acid (RNA), whether single-stranded or double-stranded, and any () chemical modifications thereof, ucn modifications include, but are not limited to, modifications at cvtosine exocvchc amines, substitution of 5-bromouracιl. backbone modifications, base analogs, methvlations, unusual base-pairing comDinations. and the like. Infra. "Phosphate ester refers to a compound having the general formula RO(PO)(OR')(OR"), where R, R' and R ' are independently selected from hydrogen, alkvl, arvl, arvlaikyl, and heteroarvl "Phosphodiester" refers to a phospnate ester in which two hvdroxvl groups of the phosphoric acid are esteπfied witn organic residues R ' O-POT H-OR " where R' and R" are independently selected from the group consisting of hydrogen, alkyl, aryl, arylalkyl, or heteroarvl Ohgonucleotides and nucleic acids are typically phosphodiesters in which the 3 ' and 5' positions ot neighboring pentose units are linked by esteπfication with a phosphate residue. "Phosphoramidate" refers to a phosphodiester in which one or more of the hvdroxyl groups is replaced with an amino group. II. lontophoretic Delivery of Nucieotide-Based Drugs A. Overview In accordance with one aspect of the present invention, novel methods for delivering nucleotide-based pharmaceutical agents across membranes, such as skin, in a controlled fashion are provided, such methods providing enhanced transdermal transport of nucleotide-based pharmaceutical agents, such as aptamers, πbozvmes, antisense compounds, and triple helix drugs, through the use of iontophoresis Nucleotides are phosphate esters ot glvcosides of heterocyclic bases and are the structural units of both ohgonucleotides and nucleic acids. Each phosphate functionality found in a nucleotide, ohgonucleotide, or nucleic acid can impart a negative charge to the molecule. For example, a single-stranded DNA of 20 nucleotides will have a molecular weight ot approximately 6500 and a net charge of -21 or approximately 1 negative charge per 325 daltons. Nucleotide-based pharmaceutical agents suitable for transdermal eiectrotransport are those which are effective at low concentrations, for example, less than 50 milligrams per dav, or are topically administered. Marked improvements in pharmaceutical agent bioavailability can be expected for those pharmaceutical agents which are poorly absorbed enterallv or undergo extensive first pass hepatic inactivation Exemplary nucleotide-based pharmaceutical agents which can be delivered by the svstem ot the present invention include* analgesics, anesthetics, antifungals, antibiotics, antnnflammatories, anthelmintics, antidotes, antiemetics, antihistamines, antihvpertensives. antimalaπais. antimicrobiaib. antiDsycnotics, antipyretics, antiseptics, antiarthritics. antituberculotics, antitussives. anπvirals cardioactive drugs, catnartics, chemotherapeutic agents, antidepressants depressants, diagnostic aids, diuretics, expectorants, h y pnotics, parasvmpathomimetics. sedatives stimulants, svmpathomimetics tranαumzers unnarv antnntectives, vasoconstrictors, vasodilators, and the like. The nucleotide-based drugs ot the present invention include aptamers, πbozvmes, antisense compounds, and triple helix drugs The nucleotide-based drugs typically mil have a molecular weight greater than about 350 and may range from between about 2 up to about 100 nucleotides, preferablv, from between about 2 to 50 nucleotides, and more preferably from between about 10 and 40 nucleotides Using the methods described herein, the eiectrotransport flux rate of DNAs has been found to be dependent upon the molecular weight of the DNA, with higher flux rates occurring with lower molecular weight DNAs However, it should be noted that a subpopulahon ot the iarger molecular weight oiigonucleotide-based drugs having a specific conformation may exist that can be transported with efficiencies comparable to those found with the smaller molecular weight drugs. Examples ot smaller nucleotide-based drugs include, but are not limited to di- and trinucleohdes, such as GS 375, a dinucieotide analog with potential therapeutic activity against the influenza virus (Gilead Sciences, Inc., Foster Citv, CA). In a particularly preferred embodiment, the nucieohde-based pharmaceutical agent will comprise an aptamer, a πbozyme, an antisense compound, or a triple helix drug B Aptamers Aptamers (or nucleic acid antibody) are single- or double-stranded DNA or single-stranded RNA molecules, typically ot about 10-50 and preferably from about 15-30 nucleotides in iength Examples ot aptamers include Gilead's anhthrombin inhibitor GS 522 and its derivatives (Gilead Science, Foster City, CA) See also Macava et al. (1993. Proc Natl Acad. Sci. USA 90:3745-9: Bock ct al (1992) Nature (London) 25_5_:564-566 and Wang et al. (1993) Biochem 32:1899-904 Aptamers bind specific molecular targets and are useful for protein epitope targeting' , especially as receptor antagonists. Generally, aptamers function by inhibiting the actions of the molecular target bv binding to the pool ot the target circulating in the blood Typically, the target will comprise a protein and the aptamer will disrupt normal protein-protein and protein-receptor interactions through competitive binding As antagonists to cell surface receptors, aptamers can exert their effects without having to penetrate the cell membrane The molecular target can comprise either an exogenous or endogenous substance Typicnlh , tor embodiments emDloving an exogenous molecular target, a non-covalentlv associated complex ot the aptamer and the molecular target will be produced and the complex will be delivered lontopnoreticallv according to the methods described herein. The dissociation constants tor these non-covalentlv associated complexes tvpicallv will be between about 10 -3 and 10" 15 , preferably between about lO -0 and 10 " 12 , and more preferabl y between about 10" 8 and 10" lϋ molar, under physiological conditions ot salt, divalent ion concentration, and temperature. Techniques tor measuring dissociation constants are well known in the art. S c, c • , PCT Publication No WO 91 /19813. Aptamers that are specific for a given biomolecuie and that can be delivered using the methods described herein can be identified by using techniques known in the art. See, e. ., Toole et al. (1992) PCT Publication No. WO 92/14843; Tuerk and Gold (1991) PCT Pubhcation No. WO 91 /19813; Weintraub and Hutchinson (1992) PCT Pubhcation No. 92/05285; and Ellington and Szostak (1990) Nature 346:818. Briefly, these techniques typically involve the complexation of the molecular target with a random mixture of ohgonucleotides. The aptamer- molecular target complex is separated from the uncompiexed ohgonucleotides. The aptamer is recovered from the separated complex and amplified. This cycle is repeated to identify those aptamer sequences with the highest affinity for the molecular target. C. Ribozvmes Ribozymes are structured RNA molecules that are capable of catalyzing a chemical reaction, such as the cleavage of a phosphodiester bond. See Symons (1992) Ann. Rev. Biochem. 71:641-671 : Kruger et al. (1982) Cull 21.147-157; Zaug and Cech (1986. Science 231:470: Cech ct al. (1982) ££1121:147-157; Altman, in "Ribonuclease P: An Enzyme with a Catalytic RNA Subunit " in Adv. Enzymol. 62, 1-36 (1989). More specifically, ribozvmes can comprise bifunctional RNA molecules containing a catalytic RNase activity as well as antisense sequences that can direct the molecule to the appropriate target mRNA. Ribozymes are typically 30-50 nucleotides in length. Ribozvmes can be readilv altered or synthesized to cleave any desired single-stranded DNA sequence, and thus can be used to target virtually any RNA transcript or to characterize DNA molecules with only few limitations on the sites that are recognized. S e. Kim ct al. (1987) Proc Natl. Acad. Sci. USA 84:8788-8792; Haseloff ct al. (1988) Nature 234:585-591; Cech (1988) 1AMA 2^0:3030-3034; Jeffries et al. (1989) Nucleic Acids Research 17:1371-1377. These nbozvme derivatives include anv RNA molecule which has the active site ot anv known nbozvme which has a deoxvnbonuclease activity. This active site may be altered to specifically cleave a desired single-stranded DNA sequence. Such an RNA molecule need onlv contain those essential portions ot the nbozvme necessary tor the deoxvnbonuclease activity Such ribozvmes can be readil \ designed bv those ot ordinary skill in the art bv use ot anv number ot standard techniques and no undue experimentation is required to determine which ot those ribozvmes are active.. Sec. e. , Cech et al PCT/US887/03161 and WO 88/04300; Lambowitz ( 1989) £__llϋ__.323; and Van der Veen ( 1986) Cell 44.225. and Murphy and Cech (1990) Proc Natl Acad. Sci USA 86:9218-9222. For example, it is well known in the art that a nbozvme can be constructed by the interaction ot two separate oligoribonucleotides, one of which is cleaved at a particular phosphodiester bond when incubated under known, appropriate conditions. Sec, e.g., Uhlenbeck (1987) Nature 328:590-600: Haseloff and Gerlach (1988) Nature 224:585-591. D. Antisense Compounds For diseases that result from the inappropriate expression ot genes, specific prevention or reduction of the expression of such genes represents an ideal therapy. In principle, production of a particular gene product may be inhibited, reduced or shut off by hybridization of a single-stranded deoxynucleotide or nbodeoxvnucleotide complementary to an accessible sequence in the mRNA, or a sequence within the transcript which is essential for pre-mRNA processing, or to a sequence within the gene itself. This paradigm for genetic control is often referred to as antisense or antigene inhibition. Antisense compounds are ohgonucleotides that are designed to bind and disable or prevent the production of the mRNA responsible for generating a particular protein. Antisense compounds include antisense RNA or DNA, single or double stranded, ohgonucleotides, or their analogs, that can hybridize specifically to individual mRNA species and prevent transcription and /or RNA processing of the mRNA species and /or translation of the encoded polypeptide and thereby effect a reduction in the amount of the respective encoded polypeptide. Ching et al. Proc. Natl. Acad. Sci. U.S.A. 86:1 006-10010 (1989); Broder et al. Ann. Int. Med. 113:604-618 (1990); Loreau et al. FEBS Letters 274:53-56 (1990); Holcenberg ct al W091 / 11535; U.S.S.N. 07/530,165 ("New human CRIPTO gene"), WO91/09865; WO91 /04753, WO90/ 13641; WO 91 /13080, WO 91 /06629, and EP 386563) Antisense compounds can provide a therapeutic function bv inhibiting in vivo the formation of one or more proteins that cause or are involved with disease. Antisense compounds complementary to certain gene messenger RNA or virai sequences have been reported to inhibit the spread ot disease related to viral and retroviral infectious agents (See. tor example, Matsukura et al (1987) Proc ι\atl Acad Sci USA 84 7706. and references cited therein) Antisense compounds ot various lengths can be delivered using the methods described herein, althougn such antisense compounds tvpicallv comprise a sequence ot at least about 15 consecutive nucleotides Examples ot antisense compounds include G 1128 (Genta. Inc., San Diego, CA), OL(l)p53 (Lynx Pharmaceuticals), Amp gen (Hemm Pharmaceuticals), Isis 1082 and Isis 2105 (Isis Pharmaceuticals, Carlsbad, CA) E. Triple Helix Drugs Ohgonucleotides also can bind to duplex DNA via triple helix formation and inhibit transcription and /or DNA synthesis. See, e.g., Maher et al. (1989) ≥α£r__£2_l__ . 725-730. Triple helix drugs (also referred to as triple strand drugs; are ohgonucleotides that bind to sequences of double-stranded DNA and are intended to inhibit selectively the transcription of disease-causing genes, such as viral genes, e.g., HIV and herpes simplex virus, and oncogenes, i.e., thev stop protein production at the cell nucleus. These drugs bind directly to the double stranded DNA in the cell's genome to form a triple helix and thus, prevents the ceil from making a target protein. See, e.g., PCT publications Nos. WO 92/10590, WO 92/09705, WO91/06626, and U.S. Patent No. 5,176,996. Typically, the triple helix drug will comprise a DNA ohgonucleotide in the range of about 20 to 40 nucleotides. F. Analogs and Derivatives The site specificity ot ohgonucleotides (e.g., aptamers, ribozymes, antisense compounds, and triple helix drugs) is not significantly affected bv modification of the phosphodiester linkage or bv chemicai modification ot the ohgonucleotide terminus. Consequently, these oligonucieotides can be chemically modified; enhancing the overall binding stability, increasing the stability with respect to chemical degradation, increasing the rate at which the ohgonucleotides are transported into cells, and conferring chemical reactivity to the molecules. The general approach to constructing various o gonucleotides useful in antisense therapy has been reviewed by vander Krol ct al. (1988) Biotechniqnes 6-958-976 and Stein et al (1988) Cancer Res 48.2659-2668 Accordingly, aptamers, ribozvmes. antisense compounds, and triple helix drugs also can include nucleotide substitutions, additions, deletions, or transpositions, so long as specific hybridization to or association with the relevant target sequence is retained as a functional property ot the ohgonucleotide For example, some embodiments will employ phosphorothioate analogs which are more resistant to degradation D\ nucleases than their naturallv occurring phospnate diester counterparts and are thus expected to have a higher oersistence in vivo and greater potency (see, e.g , Campbell ct al (1990) J Bioche Biopnvs Methods 20.259-267) Phospnoramidate derivatives ot ohgonucleotiαes also are Known to bind to complementary polvnucleotides and have the additional capability ot accommodating covalentlv attached gand species and will be amenable to the methods ot the present invenfton. See, tor example, Froehier et al. (1988) Nucleic Acids Res 16_ 11V4831 In some emoodiments the nucleotide-based pharmaceutical acent will comprise O-methvlπbonucleotides (EP Publication No 360609). Chimeπc ohgonucleotides may also be used (Dagle et al. (1990) Nucleic Acids Res 18: 4751). For some applications, the pharmaceutical agent will comprise polyaπude nucleic acids (Nielsen et al. (1991) Science 254:1497 and PCT publication No. WO 90/15065) or other catioruc derivatives (Letsmger et al. (1988) J. Am. Chem. Soc. 110:4470- 4471). Other applications mav utilize o gonucleotides wherein one or more ot the phosphodiester linkages has been substituted with an isostenc group, such as a 2-4 atom long lnternucleoside linkage as described in PCT publication Nos. WO 92/05186 and 91 /06556, or a tormacetal group (Matteucci et al. (1991) I. Am Chem SΩ__, 1127767-7768) or an amide group (Nielsen et al. (1991) ≥α__n__£ 251:1497-1500) In addition, nucleotide analogs, for example wherein the sugar or base is chemically modified, can be employed in the present invention. "Analogous" forms of puπnes and pyπmidines are those generally known in the art, many of which are used as chemotherapeutic agents. An exemplary but not exhaustive list includes 4-acetylcvtosιne, 5-(carboxyhvdroxvlmethvi) uracil, 5-fluorouracιl, 5-bromouracil, 5-carboxvmethvlammomethyl- 2-thιouracιl, 5-carboxymethvlamιnomethvluracιl, dihvdrouracil, inosine, N fa -ιsopentenvladenιne, 1-methvladenιne, 1-methvlpseudouracιl, 1-methylguanιne, 1-methvhnosιne, 2,2-dιmethyiguanιne, 2-methviadenιne. 2-methylguanιne, 3-methylcytosιne, 5-methvlcytosιne, N6-methvladenιne, 7-methvlguanιne, 5-methvlamιnomethvluracιl, 5-methoxyam_nomethyl-2-thιourac_l, β-D-mannosvlqueosine, 5 ' methoxycarbonylmethyluracιi, 5-methoxyuracιl, 2-methvlthιo-N (5 -ιsopentenvladenιne, uracιl-5-oxvacetιc acid methvlester, uracιl-5-oxyacetιc acid (v), wybutoxosine, pseudouracil, queosine, 2-thιocvtosιne, 5-methvl-2-thιouracιl, 2-thιouracιl, 4-thιouracιl, 5-methvluracιl, N-uracιi-5-oxvacetιc acid methvlester, uracιl-5-oxvacetιc acid (v ), pseudouracil, queosine. 2-thιocvtosιne, and 2,6-dιamιnopuπne In addition, the conventional bases bv halogenated bases Furthermore, the 2'-furanose position on the base can have a non-charged bulky group substitution. Examples ot non-charged bulk . croups include branched alkvls, sugars and branched sugars Terminal modification also provides a useful procedure to modit \ cell type specificity, pharmacoKinetics, nuclear permeability, and absolute cell uptake rate for ohgonucleotide pnarmaceutical agents hor example suostitutions at the 5 ana 3 ends include reactive groups which allow covalent cross nking ot ^ the nucleotide-based pharmaceutical agent to other species and bulky groups which improve cellular uptake Sec. e , Ohgodeoxynucleotides Antisense Inhibitors ot Gene Expression. (1989) Cohen, Ed., CRC Press, Prospects tor Antisense Nucleic Acid Therapeutics for Lancer and AIDS. (1991 ), Wickstrom, Ed , Wilev-Liss, Gene Regulation Biology of Antisense RNA and DNA. (1992) 0 Eπckson and Izant, Eds., Raven Press; and Antisense RNA and DNA. (1992), Murray, Ed , Wilev-Liss For general methods relating to antisense compounds, see Antisense RNA and DNA, (1988), D.A Melton, Ed , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) G Secondary Structure of DNA Polvnucleotide chains contain several bonds about which there is tree rotation In the absence ot anv intrastrand interactions each monomer would be tree to rotate with respect to its adjacent monomers The three-dimensional configuration of such a chain is called a random coil, it is a somewhat compact and globular structure that changes shape continually However, tew nucleic acid molecules exist as random coils due to the many interactions (e g , hydrogen bonds, hydrophobic interactions, ionic bonds, van der Waais interactions, and disulfide bonds) between the elements of the chain DNA structure is verv rich in variety Although the double-stranded B helix is the predominant form, additional helical and other forms exist, including the A hehx, the Z helix, circular, and superhehcal DNA Additionally, tRNA is composed of single strands ot πbonucleic acid containing segments that have a high degree of base complentaπtv with downstream segments which allows intrastrand base pairing and the formation of loops Consequently, tRNA contains a high degree ot secondary structure consisting ot base-paired double- helical segments, resembling a clover leaf, in which the tour stems represent double-stranded helical segments composed of complementarv base sequences and hydrogen-bonded bases Although not wishing to be bound bv the following theory, bv analogy with polvacrvlamide and agarose gel electrophoresis. the conformation or the o gonucieotide-based pharmaceutical agent might be expected to have an effect on the electrotransport rate ot the ohgonucleotide with the more compact ohgonucleotide more easily traversing the skin as compared with the floppy conformation ot an extended conformation Conversely, extended conformations mignt be snaking their vv av tnrougn channels in the stratum corneum w hile the transport ot the compact or contormational restricted ohgonucleotides ma y be retarded due to their relatively larger cross-sectionai areas This conformation effect could be important, since aptamers have been suggested to adopt tightlv-tolded structures ι w Wans: ct al (1993) Biochemistry 32 1899-1904), whereas rioozvmes tvpicailv will consist ot both selt- complementarv hairpin catalytic as well as unstructured, single-stranded antisense domain (see Zaug ct al (1986) Nature 324 429-433), and in contrast, individual antisense compounds typically will posses no innerent seconαarv structure Thus these nucleotide-based pharmaceutical agents conceivably could exhibit different electrotransport behavior depending on their conformation Surprisingly, we have found that the conformation of the ohgonucleotide did not seem to have anv appreciable effect on electrotransport flux rate. These results are shown in Fig. 6 EQ. Compositions of Nucleotide-based Pharmaceuticals As applied to the lontophoretic delivery ot nucleotide-based pharmaceutical agent, the invention provides nucleotide-based pharmaceutical agents with a charge-to-mass ratio that allows the pharmaceutical agent to be delivered in therapeutically effective amounts. Typically, the charge-to-mass ratio of such a compositions will exceed one charge per 5000 daltons, and more typically, one charge per 2500 daltons Preferably, the charge-to-mass ratio will be equal to or exceed one charge per 1000 daltons, more preferably, one charge per 500 daltons . The nucleotide-based pharmaceutical agent can be admixed with an acceptable physiological carrier solution, such as water, aqueous alcohols, propylene glycol, and dimethvlsultoxide, to make a composition suitable tor dermal contact and lontophoretic delivery Acceptable physiological carrier" includes those solutions which do not intertere with the effectiveness or the biological activity of the active ingredients and which are not toxic to the hosts to which it is administered Well known techniques tor choosing appropriate carriers and formulating the proper mixtures are exemplified in Banga et al. supra, Lattin et al. (1991) Ann. N.Y. Acad. Sci., 618:450; and Remington s Pharmaceutical Science, 15th Ed., Mack Publishing Company, Easton, Pennsylvania. (1980), which are incorporated herein bv reference Typically, the carrier solution will also contain other ionic species, in addition to the o gonucleotides For example, these ionic species can arise from buffer solutions that mav be present to maintain the pH of the solution As expected from a coulombic mechanism ot electrotransport, to achieve the highest transport efficiency, the concentration of all ionic species, save the ohgonucleotide or DNA itself, should be minimized More specifically, we have found that increasing the salt concentration in the carrier solution results in a net decrease in the electrotransport rate of the DNA These results are shown in Fig i According to some embodiments, Lιpotectιn I (available from BRL, Gaithersburg, Md., see Feigner ct al (1987) Proc Natl Acad. Sci USA 84 7413). DOTMA (N-[l-(2,3-dιolevloxy)propyl] -N,N,N-trιmethvlammonιum chloride ) will be utilized in combination with the nucleotide-based pharmaceutical agent. Lιpotectιn 1M is a posome tormulahon ot a cationic and a neutral hpid that interacts with DNA to form a pid-DNA complex. See. e.g , Feigner et al. (1989) Nature 337:387. The fusion of the hpid-DNA complex with cells results apparently in efficient transfer of the DNA into the cell and is commonly used to increase the transfection efficiency ot mammalian cells in culture. Surprisingly, we have found that the addition of small amounts of Lipotectin™ to DNAs enhanced the electrotransport of the DNAs slightly. These results are shown in Fig. 4. According to other embodiments, other posomal formulations, tor example, those comprising 3-β-[N-(N', N'-dιmethyl-amιnoethane)-carbamoyl] cholesterol in combination with dioleoylphosphatidylethanoiamine (see Gao and Huang (1991) Biochem. Biophvs Res. Cnmmun. 179:280-5): or popoiylysines (i.e., low molecular weight (Mr approximately 3000) poly(L-lysιne) (PLL) conjugated to N-glutarylphosphatidylethanolamine and containing an average of two phospholipid groups per molecule of PLL, see Zhou et al. (1991) Biochim. Biophvs Acta 1065:8-14) will be used. The hposomal formulations also can comprise pH-sensitive liposomes (e.g., composed of cholesteryl hemisuccinate and dioleoylphosphatidylethanolamine at a 2.1 molar ratio) or non-pH-sensitive liposomes. When Lιpofectιn r or another hposomal formulation is used in combination with the compositions and methods described herein, the additive will typically be present in the donor reservoir in an amount between about 1 and 100 μg/ml, preferably, between about 2 and 50 μg/ml, and more preferably, between about 2 and 25 μg/ml. In addition to the nucleotide-based pharmaceutical agents, the composition can contain other materials such as dyes, pigments, inert fillers, or other permeation enhancers, excipients, and conventional components of pharmaceutical products and transdermal therapeutic systems known in the art. Thus, according to some embodiments ot this invention, chemical enhancers (i.e., penetration or permeation enhancers) will be incorporated into the donor reservoir of the lontophoretic device and utilized to alter the lontophoretic transport rate. For example, the coapphcation ot oleic acid to the skin causes a large decrease in the skin impeαance or resistance which is inversely related to permeability or transport. See Potts ct al. (1992) Solid State Ionics 53-56:165-169 Thus, instead ot the current passing primarily tnrougn the snunt pathways le.g , the follicles and sweat ducts ) the ions le g , the nucleotide-based p harmaceutical agent) constituting the current can more uniforml y permeate the hpid milieu of the stratum corneum at a lower current αensitv Alternatively, the use or chemical enhancers will allow tor an increased rate ot lontophoretic transport ot *> the nucleotide-based pharmaceutical agent as compared to the transport rate tound at the same current density in the absence of the chemical enhancer Using the compositions and methods described herein, the electrotransport flux rate ot the nucleotide-based pharmaceutical agents has been tound to increase in a concentration-αependent manner These results are snown 0 in Fig 3 Generally, the nucleotide-based pharmaceutical agent will be present in the reservoir of the iontophoresis device in an amount ranging from between about 1 to about 500 mg/ml, and preferably from between about 5 to about 200 mg/ml and the device will be capable of delivering between about 0.1 and 20 mg, and preferably between about 0 1 and 10 mg, ot the nucleotide-based pharmaceutical agent during a 24 hour period for an iontophoresis device having about a 20 cm 1 donor reservoir IV In Vitro Testing of Nucieotide-Based Pharmaceutical Agents The m vitro skin permeation rate of a pharmaceutical agent can be measured using diffusion cells Human, mouse or porcine skin is placed on the lower half of the diffusion cell with the stratum corneum facing the donor compartment The donor compartment contains a solution of the pharmaceutical agent and the cathode The receiver compartment contains a buffer solution and the anode. An electric current is applied and the amount ot transported drug can be calculated (μg/cm^ hr) Alternatively, an iontophoresis device containing the pharmaceutical agent to be tested can be placed on the stratum corneum The receiver compartment again would contain a buffer solution The device is activated and the amount ot transported drug can be calculated (μg/cm-^ hr) Conventional flow-through diffusion cells can also be used to measure the in vitro skin permeation rate of pharmaceutical agents Typically these cells will have an active area of 1 cm 1 * and a receiving volume of 3 mi The receptor fluid, generally isotonic saline or buffer solution, is pumped into and through the cells, bv a peristaltic pump Samples can be collected in glass \ lais arranged in an automatic traction collector The amount ot drug permeating across the skin (μg/cm-- hr) is calculated from the cumulative release The electrotransport behavior ot a pharmaceutical agent can also be assessed using the conventional analytical techniques and gel or capiilar \ electrophoresis Preparation measurements mav also be performed on excised --kin in conventional ditrusion ceil tests ^ e e e , Lattin ct al -.itjira V !π V ivo Delivery ot Nucieotide-Based Pharmaceutical Agents A Therapeutic Indications The compositions and methods described herein will find use in the treatment ot a variety ot diseases, including but not limited to those described below The compositions and methods describeα herein can be utilized tor either the svstemic or local administration ot the nucleotide-based pharmaceutical agent, depenαing on the therapeutic indication For example, nucleotide-based pharmaceutical agents can be designed to prevent expression of diverse potential target genes, including oncogenes, fungal genes, and any other gene known to be activated specifically in the skin. These pharmaceutical agents can then be delivered directly to melanomas, Kaposi sarcomas, psoriasis lesions, and fungal infected skin, and the like. The compositions and methods described herein will find use in the treatment ot viral, fungal, and bacterial infections ot the skin and mucous membranes, including genital warts caused by the human papilloma virus and infections caused by Herpes viruses. In addition, the present invention provides for the delivery of therapeutic compositions of nucleotide-based pharmaceutical agents directly to block mediators of inflammation, including cytokines, growth factors, cell adhesion molecules or their gands and receptors thereof, as well as key enzymes in pathways leading to inflammation. More specifically, these blocking actions include preventing the expression ot cvtokines (such as IL-1), growth factors (such as TGF-α and EGF), or cell adhesion molecules (such as ELAM and ICAM); or the receptors for cytokines (such as IL-1), growth factors, or cell adhesion molecules. Key enzvmes whose expression may be blocked include protein kinase C and phospho pase A or C. Thus, according to one aspect ot this invention, topical or transdermal formulations of the complexes described herein are applied directly knees or other joints to alleviate inflammation and the like. In yet another aspect, the compositions and methods described herein can be used to treat conditions in which improper immune or inflammatory responses have been implicated such as psoriasis (e.g., by blocking the expression of I -1, TGF-α, amphireguhn, or IL-6); atopic dermatitis and eczema (e.g., by blocking the overexpression ot IgE); rheumatoid arthritis; allergic rhinitis (e.g , bv blocking the expression of IL- ); and the like In an additional aspect, the compositions and methods described herein can be used to treat certain cancers ot the skin and mucous membranes, such as melanoma, mvcosis rungoides, and squamous cell carcinoma (including of the cerv'ix ), tor example, bv blocking the expression ot certain factors which promote cell growth and/ or adhesion and wruch are Deiieveα to be involved in metastasis The compositions ana methods described herein also can be employed tor transαer al genoculation ' Sec. Watanabe ct al. ( 1993) Proc N'atl Acad Sci 20:4523-4527 and Bioworld Today, ' "Genoculation Induces Immune Responses in Mice", Mav 17. 1993 More specifically, a nost suffering from advanced melanoma have been innocuiated with an incompatible human major histocompatibilitv complex (MHC) gene that has been incorporated within a hposome envelope. In BALB/C mice with induced forms ot colon adenocarcinoma or tibrosarcoma, pretreanng the animals with the MHC gene a few days before inducing tumors was found to double survival time. This "genoculation" is thought to induce a cytotoxic T cell response to the MHC tag, and more importantly, to rumor antigens that the immune system normally does not see and that prevent the body's immune response from recognizing and destroying the malignant tissue. Thus, one aspect ot the present invention provides tor the lontopnoretic delivery of MHC genes to llhct an immune response. B. Dosages According to this invention, a therapeutically or pharmaceutically effective amount of a nucleotide-based pharmaceutical agent is delivered lontophoretically to a patient in need of such an agent. The compositions and methods described herein can be employed for the prophylactic and/or therapeutic treatments. In therapeutic applications, the compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish thus is defined as "therapeutically effective amount or dose. ' Amounts effective tor this use will depend on the seveπtv and course of the disease, previous therapy, the patient s health status and response to the drugs, and the judgment of the treating physician. For standard dosages of conventional nucleotide-based pharmaceutical agents, see, e.g., Physicians Desk Reference (1992 Edition); and American Medical Association (1992) Drug Evaluations Subscriptions. In prophylactic applications, the nucieotide-based pharmaceutical agent is administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a "prophvlacticallv effective amount or dose." In this use, the precise amounts again depenα on tne patient s state ot health, weight, and the like. Once improvement ot the patient s conditions has occurred, a maintenance dose can be administered it necessary Subsequently, the dosage or the frequency ot administration, or both, can be reduced, as a function of the symptoms, to a le . ei at wnich the improved condition l.*** retained When the symptoms nave been mted to the desired level, treatment can cease Patients can. however, require intermittent treatment on a lυn^-term basis upon am recurrence ot the disease symptoms In general, a suitable effective dose of the nucleotide-based pharmaceutical agent which can be delivered lontopnoreticallv according to the methods described herein will be in an amount ranging trom between about 0.1 to about 10 milligram (mg) per recipient per dav using an iontophoresis device having a 20 cm'- donor reservoir and a current ot less than aDout 0.5 mAmps/ cm-, preferably in the range of between about 0 5 to about 5 mg per dav, and most preferablv in an amount ot about 0.5 to about 1 mg. C. lontophoretic Delivery The nucleotide-based pharmaceutical agents described herein can be administered transdermallv using iontophoresis. This form ot administration typicallv involves the delivery ot a pharmaceutical agent tor percutaneous passage of the drug into the systemic circulation of the patient. However, the nucleotide- based pharmaceutical agents also can be delivered directly to pathological or diseased tissue using lontopnoresis for the local administration of the analog. The skin sites include anatomic regions for transdermallv administering the drug as represented by the forearm, abdomen, chest, back, buttock, mastoidal area and the like. The therapeutic composition can be delivered bv a standard lontophoretic device. In general, iontophoresis is an introduction, by means of electric current, ot ions of soluble salts into the tissues of the body. More specifically, iontophoresis is a process and technique which involves the transfer ot ionic (charged) species into a tissue (tor example through the skin ot a patient) by the passage of a electric current through an electrolyte solution containing ionic molecules to be delivered (or precursors for those ions), upon application ot an appropriate electrode polarity That is, ions are transferred into the tissue, trom an electrolyte reservoir, by application ot electromotive force to the electrolyte reservoir. In lontophoretic systems, the rate of release is primarily controlled bv the voltage or current A wide variety ol iontophoresis devices are presently known. See. e < ., Phipps ct al U.S. Patent No 4.744.788, Phipps et al U.S. Patent No 4,747,819, Tapper ct al. European Patent Application Publication No 031S776, Jacobsen ct al European Patent Application Publication No 0299631. Petelenz ct al U S Patent No. 4.752.285; Sanderson et al U S Patent No 4,722,726; Phipps et al. U.S Patent No. 5,125,894; and Parsi U S Patent No 4.731.049. Badzinski a al (1993) U.S. Patent No. 5,207,752; Gvorv ct al (1993) U S Patent No 5,203,768; Gvorv ct al (1992) U.S Patent No 5.162.042, Phipps ( 1 L) 2 ) PCT Publication No WO 92/ 17239. anαrau ct .(.'. (1992) TCT Pubhcation No. WO 92/ 15365; Gvorv et al. ( 1992 ) Canadian Patent Publication 2.042,994; Gvorv et al. (1992) U.S. Patent No. 5.158,537, Gvorv < • : al. (1992) PCT Publication No. WO 92/07618; Myers et al. 1 1992) U.S. Patent No. 5,147.297; Gvorv ct al. (1992) U.S. Patent No. 5.147.297, Gvorv ct al. ( 1991 ) Canadian Patent Pubhcation No. 2,015,597; Gvorv ft al. ( 1992) U.S. Patent No. 5,084,006; Gvorv ct al. U.S. Patent No. 5,162,043; Haak ct al. ( 1992) U.S. Patent No. 5,167,616, Gvorv ct al. (1990) PCT Publication No. 90/09413; Theeuwes et al. (1992) U.S. Patent No 5.080,646; Theeuwes et al. (1992) U.S. Patent No. 5,147,296; Theeuwes et al ( 1992) U.S. Patent No. 5.169,382; Theeuwes et al. (1992) U.S. Patent No. 5,169,383; Theeuwes (1990) U.S. Patent No. 4,978,337; Moodie et al. (1992) U.S. Patent No. 5,125.894; Haak et al. (1990) U.S. Patent No. 4,927,408; the full disclosures of each which are incorporated herein by reference. In typical, conventional, electrotransport or iontophoresis devices, two electrodes are generally used. Both electrodes are disposed so as to be in intimate electrical contact with some portion (typically skin) of the subiect (human or animal) tvpicailv by means of two remote electrolyte-containing reservoirs, between which current passes as it moves between the skin and the electrodes. Generally the active electrode includes the therapeutic species as a charged ion, or a precursor tor the charged ion, and the transport occurs through application of the electromotive force to the charged therapeutic species. An appropriate potential is initiated between two electrode systems (anode and cathode) in electrical contact with the skin. If a positively charged drug is to be delivered through the skin, an appropriate electromotive force can be generated by orienting the positively charged drug species at a reservoir associated with the anode. Similarly, it the ion to be transferred across the skin is negatively charged, appropriate electromotive force can be generated by positioning the drug in a reservoir at the cathode. Of course, a single system can be utilized to transfer both positively charged and negatively charged drugs into a patient at a given time; and, more than one cathodic drug and /or more than one anodic drug may be delivered from a single system duπng a selected operation. In conjunction with the patient ' s skin in electrical communication with the electrodes, the circuit is completed bv connection of the two electrodes to a source ot electrical energy as a direct current; for example, a batterv or a source ot appropriately modified alternating current. For general discussions oi iontophoresis, see, c.<-.. Tyle ( 1989) 1 Pharm. Sci. 75:318: Burnette. Itmtoviwrcsis (Chapter 11) in Transdermal Drug Delivery Hadgratt and Guv (eds.) Marcel Dekker, Inc.: New York. NY; Phipps et al. (1988) Solid State Ionics 28-30:1778-1783: Phipps et al. ( 1989) 1 Pharm. Sciences 78:365-369: and Chien ct al. (1988) 1 Controlled Release 7:1-24. the full disclosures ot which are incorporated herein bv reference. Electrode refers to an electrically conductive member tnrougn whicn a current passes duπng operation A variety of electrode materials, ranging trom platinum to silver-silver chloride, are available tor these devices The primary difference in these materials is not in their ability to generate an electric 5 potential across the skin, but rather in certain nuances associated with their performance ot this function. For example, platinum electrodes hvdroivze water, thus liberating hydrogen ions and subsequently, changes in pH. Obviously, cnanges in pH can influence the lonization state ot therapeutic agents and their resulting rate ot lontophoretic transport. Silver-silver chloride electrodes, on the l 0 other hand, do not hvdroivze water. However, these electrodes require the presence of chloride ion which may compete tor current-induced transport. Electrotransport devices generally require a reservoir as a source of the species (or a precursor ot such species) which is to be moved or introduced into the bodv. The reservoir tvpicailv will comprise a pool ot electrolyte solution, tor 1 5 example an aqueous electrolyte solution or a hvdrophilic, electrolyte-containing, gel or gel matrix, semi-solid, foam, or absorbent material. Such pharmaceutical agent reservoirs, when electrically connected to the anode or the cathode ot an iontophoresis device, provide a source of one or more ionic species for electrotransport. 2 0 Manv iontophoresis devices employ a selectively permeable membrane The composition of this membrane will vary with the particular needs of the system and will depend upon the composition ot the electrolyte reservoir, i.e., the nature of the pharmaceutical agent, the transference of current out of the reservoir, and the desired selectivity to transport of particular types of 2 5 charged and uncharged species. A microporous polvmer or hvdrogel such as is known in the art can be utilized. See. e.g., U.S. Patent No. 4„927,408. Suitable permeable membrane materials can be selected based on the desired degree of permeability, the nature of the complex, and the mechanical considerations related to constructing the device. Exemplary permeable 3 0 membrane materials include a wide variety of natural and synthetic polymers, such as polydimethylsiloxanes (silicone rubbers), ethvlenevinvlacetate copolvmer (EVA), polvurethanes, polvurethane-polvether copolvmers, polvethvlenes, polvamides, polvvinvlchloπdes (PVC), polypropyienes, polycarbonates, polvtetratluoroethvlenes (PTFE), celluiosic materials, e.g , cellulose triacetate and ^ 5 cellulose nitrate / acetate, and hvdrogels, e.g , 2-hvoroxvethvlmethacrvlate (HEMA) Generally, butters will also be incorporated into the reservoir to maintain the reservoir environment at the same charge as the electrode Typicaih , to minimize competition for the electric current, a butter naving the opposite charge to the drug will be employed In some situations, tor example, 0 wnen tne appropriate salt is used, tne drug mav act as its own butter Other variables which may effect the rate ot transport include dru_r concentration, buffer concentration, ionic strength, nonaoueous cosoivent.. , and anv other constituents in the formulation. However, as discussed above, to achieve the highest transport efficiency, the concentration ot all ionic species, save tne pharmaceutical asent itself, is minimized. The backing or enclosure of the drug de verv system is intended primarily as a mechanical support tor the reservoir or matrix, in the simplest case, the matrix is exposed directlv to the skin or membrane ot the host, and the backing is a strip or patch capable of being secured to the skin, typically with the matrix acting as an adhesive. In such constructions, the backing will usually be impermeable to the complex. This lmpermeabiiitv inhibits the ioss of the complex. Suitable backing materials will generally be thin, flexible films or fabrics such as woven and non-woven fabrics and polymeric films, such as polyethylene, polypropylene, and silicone rubber; metal films and foils; and the like. The delivery device can be held in place with the adhesive ot the matrix, with an adhesive along the perimeter of the matrix, with tape or elastic, or any other means, so long as the device allows the pharmaceutical agent to be transported through the skin. The device can be placed on any portion of the skin or dermal surface, such as the arm, abdomen, thigh, and the like. Furthermore, the device can be in various shapes and can consist ot one or more complexes and /or transport areas. Other items can be contained in the device, such as other conventional components of therapeutic products, depending upon the desired device characteristics. In the conventional topical treatment by iontophoresis, the direct current is applied through moist pad-type electrodes with size corresponding to that of the skin region to be treated. The interposition ot a moist pad between the electrode plate and the skin is necessary tor making a perfect contact, preventing any skin burns, overcoming skin resistance, and protecting the skin from absorbing any caustic metal compounds formed on the metal plate surface. The drug is administered through an electrode having the same charge as the drug, and a return electrode opposite in charge to the drug is placed at a neutral site on the body surface. The operator then selects a current intensity below the pain threshold level of the patient and allows the current to flow for an appropriate length ot time. Ions transferred through the skin are taken up by the micro-circulation at the dermal-epioermal lunction, while the current proceeds through the skin tissues to the return electrode. The current intensity should be increased slowly, maintained for the length ot time ot the treatment, and then decreased slowly at the end of the treatment. The current must be within comfortable toleration of the patient, with a current densitv which is generally less than 0.5 mAmp/ cm^ of the electrode surface. The therapeutic composition can be delivered bv a standard lontophoretic device Owing to differences in available lontopnoretic devices the procedure tor use can vary The manufacturer s instructions snould be followed tor appropriate pharmaceutical agent deliverv Bodv fluid or blood levels ot the uncomplexed pharmaceutical agent will be determined to measure the effectiveness ot the transport and bioconversion One aspect of this invention provides for the delivery of therapeutic compositions of nucleotide-based pharmaceutical agents directly to pathological or diseased tissue for the local administration of the nucleotide-based pharmaceutical agent. Nucleotide-based pharmaceutical agents can be designed to prevent expression of diverse potential target genes, including oncogenes, fungal genes, and anv other gene known to be activated specifically in the skin. These pharmaceutical agents can then be delivered directly to melanomas, Kaposi sarcomas, psoriasis lesions, and fungal infected skin, and the like using the methods described herein. In addition, the present invention further provides for the delivery of therapeutic compositions of nucleotide-based pharmaceutical agents directly to knees or other joints to alleviate inflammation and the like The invention will be more fullv described and understood with reference to the following examples These examples are provided bv wav of illustration only and not bv wav of limitation Those skilled in the art will readilv appreciate a variety of noncπtical parameters which could be changed or modified to yield essentially similar results EXPERIMENTAL MATERIALS O gonucleotides were obtained commercially or svnthesized using n commercially-available ohgonucleotide synthesizer (e g , Applied Biosvstems Model 394 O gonucieotide Synthesizer) and cvanoethvi phosphor amidite chemistry The DNAs were end-labeled with 3 p and T4 polynucleotide kinase and rigorously purified from unincorporated ATP bv reverse phase chromatography In addition, collections of radioactivelv-labeled, singie-stranded ohgonucleotides and nucleic acids of random sizes can be generated using techniques well known in the art. For example, multiple rounds of DNA synthesis from a DNA template using Taq DNA polymerase, dideoxvnucleotide tπphosphates, and either ^ 2 P-labeled ohgonucieotide primers or 3 p 33 ^ or 35s_ labeled deoxvnucleosides can be performed. See Promega Protocols and Applications Guide. 2nd Ed., Promega Corp., Madison, WI (1991). In addition, any of a variety of other methodologies can be used, including Bal 31 nuclease digestion of DNA followed by radioactive labeling, "nick translation" or "random primer synthesis", which uses Dnase 1 or random ohgonucleotide primers, respectively, to create pnmer-tempiate junctions for the incorporation of radioactivelv-labeled deoxvnucleosides by DNA polymerases, etc. The labeled DNA's should be in sufficient molar excess over their templates, as well as devoid of detectable secondary structures (unless engineered into the template sequence), to ensure that no higher order, macromolecular structures are formed. The size distribution of a sample of a mixture of labeled fragments can be assessed by electrophoresis using a standard DNA sequencing gel and autoradiography. See. e.g., Sambrook et al. Molecular Cloning. Typically, a distribution ot uniformly labeled fragments extending from approximately 5-200 nucleotides is created. Synthetic membrane, hairless guinea pig, hairless mouse, or human (either living or cadaverous) skin can be prepared by techniques known in the art Although the experiments described herein employed custom-made Teflon diffusion cells, one of skill in the art will appreciate that the methods can be easily modified for use with conventional, commercially available diffusion cells A random 20-mer was shown to be resistant to degradation bv either the dermal or epidermal surfaces ot freshly-prepared hairless mouse skin in vitro duπne a 6 hour incubation in PBS at room temperature A random 20-mer that had been lontophoresed through hairless mouse skin in vitro was shown to be intact as judged bv polvacrylamide gel electrophoresis followed bv autoradiography and comparison with control DNA standards EXAMPLE 1 GENERAL METHODS Hairless mouse skin was inserted into custom-made Teflon cells possessing 0 2 ml donor and receptor volumes, at 0 3 mAmps constant current /cm^ hairless mouse skin at room temperature The compound to be tested was placed in an appropriate buffer in a donor chamber on the exterior side of the skin or membrane A "counter chamber containing suitable buffer is placed on the interior side of the skin or memorane. The donor chamber contained a silver chloride (AgCl) cathode and the receptor chamber contained a silver (Ag) anode. An experiment was performed in which DNA delivery from the anode was attempted. However, no measurable flux rate was found. Likewise, a passive transport experiment in which no current was applied was performed. Again, no measurable flux of DNA was tound. Following application ot electric current, a sample is withdrawn trom the counter chamber and analyzed. Sampling was performed by first mixing the buffer within the desired cell, removing 0.025 ml of solution, and immediately replacing it with 0.025 ml of fresh buffer. Except where noted, all donor cells contained 30 millimolar fmM) sodium chloride (NaCl), 50 mM MOPS, pH 6., and 50 mg/ml of the 3 P-labeled ohgonucleotide and the receptors contained the same, minus the ohgonucleotide. The extent of DNA transport was determined following scintillation counting using the values of specific activity calculated following the kinase end- labeling of the ohgonucleotides All flux experiments were performed in triplicate and the average values were plotted. Transport can also be assessed using an antibodv-mediated reaction, an activity assay, or bv radioachvely prelabehng the test compound, either enzvmaticaliv or metabo callv, and monitoring the radioactivity. EXAMPLE 2 EFFECT OF SALT CONCENTRATION The electrotransport of random 20-mer DNAs at three different NaCl (6 mM, 30 mM, and 150 mM) concentrations was tested The donor and receptor cells contained equivalent amounts of NaCl. As expected from a coulombic mechanism ot electrotransport, in which the DNA itself is transported bv virtue ot its actually carrying the current ( as opposed to a passive electroosmotic mechanism in which the DNA is carried along with the flow of solvent), increasing the salt concentration and therebv decreasing the fractional contribution ot the DNA to the current conductance resulted in a net decrease in DNA flux At the lowest NaCl concentration tested (6 mM), the DNA flux dropped dramatically following the 4 hour timepoint. presumaolv due to ion depletion in the receptor cell These results are shown in Fig 1 EXAMPLE 3 EFFECT OF OLIGONUCLEOTIDE SIZE The electrotransport flux rates for three difterent sizes ot random DNA (20-mer, 30-mer, and 40-mer) were determined. While the charge densities tor each ot the three size classes of DNA were virtually identical, the DNAs ranged in molecular weight from 6500 to 13,000 daltons. The flux rate was found to decrease rapidly with increase in molecular weight, with the 40-mer transporting at approximately one-fifth the rate of the 20-mer. These results are shown in Fig. EXAMPLE 4 EFFECT OF OLIGONUCLEOTIDE CONCENTRATION The electrotransport flux rates of random 20-mers at three different donor DNA concentrations (5 mg/ml, 50 mg/ml. and 200 mg/ml) were measured. The flux rate was found to increase in a concentration-dependent manner with the highest concentration tested (200 mg/ml) appearing to be sub-saturating. This experiment was done at 150 mM NaCl concentration. If corrected to account for the 30 mM NaCl concentration, flux rates in excess of 1 mg/cm^Ohr would be expected for a donor concentration of 200 mg/ml of random 20-mer. These results are shown in Fig. 3. EXAMPLE 5 EFFECT OF LIPOFECTIN™ CONCENTRATION [l-(2,3,-dιolevloxy ' propyl]-N,N,N-tπethviammonιum chloride, which is commercially available as Lipofectin™, is a hposome formulation of a catioruc and a neutral lipid that interacts with DNA to forma hpid-DNA complex. See, e.g., Feigner et al. (1989. Nature 337:387. Much as liposomes have been used as delivery vehicles for passive transdermal drug delivery, we believed that complexation of the DNA with the Lipofectin™ might render the complex more accessible to hydrophobic pathways in the stratum corneum, thus resulting in an enhancement of flux. Alternatively, the Lipofectin rM mav be acting as a permeability enhancer directlv. Lipofectin I M was added to a random DNA 20-mer in the donor cell (0 mg/ml, 2 mg/ml, and 20 mg/ml). It was found that Lipofectin™ enhanced the electrotransport of DNA s ghtlv. These results are shown in Fig 4. EXAMPLE 6 EFFECT OF DNA CONFORMATION The electrotransport flux rate of the 15-nucleotιde thrombin aptamer d(GGTTGGTGTGGTTGG) SEQ ID NO:l (see Bock et al. (1992) Nature 355:564-566. was compared to that of a random DNA 20-mer. The structure of the aptamer, consistmg of a pair of stacked G tetrads and 3 loops, has been deduced by NMR analysis and is shown in Fig. 5. The conformation of the DNA did not seem to have any appreciable effect on electrotransport flux rate. These results are shown in Fig. 6. It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The invention has been described above in some detail for the purposes of clarity and understanding. The disclosures of all articles and references, including patent publications, are incorporated herein by reference. Changes and modifications can be practiced within the scope of the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Affymax Technologies N.V. (ii) TITLE OF INVENTION: Compositions and Methods for Transdermal Drug Delivery (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Affymax Technologies N.V. , Administrative Offices (B) STREET: 4001 Miranda Avenue (C) CITY: Palo Alto (D) STATE: California (E) COUNTRY: USA (F) ZIP: 94304 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: WO (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/107,329 (B) FILING DATE: 16-AUG-1993 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/077,786 (B) FILING DATE: 14-JUN-1993 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/009,463 (B) FILING DATE: 27-JAN-1993 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/077,296 (B) FILING DATE: 14-JUN-1993 (vϋi) ATTORNE /AGENT INFORMATION: (A) NAME: Stevens, Lauren L. (B) REGISTRATION NUMBER: 36,691 (C) REFERENCE/DOCKET NUMBER: 11509-78-3/1031.2 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-496-2300 (B) TELEFAX: 415-424-0832 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (oligonucleotide) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l GGTTGGTGTG GTTGG 15

Claims: WHAT IS CLAIMED 15: 1. A method for the delivery of a pharmaceutical agent in a therapeutically effective dose to a patient, said method comprising the steps of: contacting the skin of the patient with a therapeutically effective amount of a pharmaceutical agent in an iontophoresis device, wherein the pharmaceutical agent comprises an oligonucleotide or a nucleic acid and the pharmaceutical agent is ionically charged; and applying an electric field to the interface of the device and the skin, such that the electric field transdermally delivers the pharmaceutical agent. 2. The method of Claim 1 wherein the pharmaceutical agent in an iontophoretic device is applied directly to pathoiogicai tissue or an area of localized inflammation. 3. The method of Claim 1 wherein the pharmaceutical agent comprises an oligonucleotide or nucleic acid having between 2 and 100 nucleotides 4. The method of Claim 3 wherein the pharmaceutical agent comprises an oligonucleotide or nucleic acid having between 15 and 50 nucleotides. 5. The method of Claim 3 wherein the pharmaceutical agent comprises an aptamer, a ribozyme, an antisense compound or a triple helix drug. 6. The method of Claim 5 wherein the pharmaceutical agent comprises the aptamer d(GGTTGGTGTGGTTGG). 7. The method of Claim 1 wherein the pharmaceutical agent in the iontophoresis device further comprises Lipofectin™, wherein the Lipofectin™ is present in an amount sufficient to enhance the rate of transport of the pharmaceutical agent as compared to the rate of transport of the pharmaceutical agent in the absence of the Lipofectin™. 8. The method of Claim 1 wherein an electric field of between about 0.1 and 0.5 mAmp/cm^ is applied. 9. The method of Claim 1 wherein the pharmaceutical agent is transdermally delivered in an amount ranging from between 0.1 and 10 mg per 24 hours using a iontophoresis device having a 20 cm?- donor reservoir. 10. A system for delivering a nucleotide-based pharmaceutical agent comprising: (a) a source of the nucleotide-based pharmaceutical agent to be delivered through a selected intact area of skin or mucosal tissue; and (b) an iontophoretic device containing said source. 11. The system of Claim 10 wherein the pharmaceutical agent comprises an oligonucleotide or nucleic acid having between 2 and 100 nucleotides 12. The system of Claim 11 wherein the pharmaceutical agent comprises an oligonucleotide or nucleic acid having between 15 and 50 nucleotides. 13. The system of Claim 10 wherein the pharmaceutical agent comprises an aptamer, a ribozyme, an antisense compound or a triple helix drug. 14. The system of Claim 13 wherein the pharmaceutical agent comprises the aptamer d(GGTTGGTGTGGTTGG). 15. The system of Claim 10 wherein the system further comprises Lipofectin™, wherein the Lipofectin™ is present in an amount sufficient to enhance the rate of transport of the pharmaceutical agent as compared to the rate of transport of the pharmaceutical agent in the absence of the Lipofectin™. 16. The system of Claim 10 wherein the iontophoretic device is capable of applying an electric field of between about 0.1 and 0.5 mAmp/cmA 17. The system of Claim 10 wherein the pharmaceutical agent is transdermally delivered in an amount ranging from between 0.1 and 10 mg per 24 hours using a iontophoresis device having a 20 cm^ donor reservoir.

Inventors: OLDENBURG KEVIN R, SELICK HAROLD E, OLDENBURG, KEVIN, R., SELICK, HAROLD, E.
Applicants: AFFYMAX TECH NV, OLDENBURG KEVIN R, SELICK HAROLD E, AFFYMAX TECHNOLOGIES N.V., OLDENBURG, KEVIN, R., SELICK, HAROLD, E.
Application year: 1994
Main classification: A61K31
IPC codes: A61K9, A61K47, A61K48, A61N1, C07H21, C12N15